We propose to study the effects of protein alkylation on the structure, dynamics and folding of thioredoxin which catalyzes dithiol-disulfide exchanges reactions. A member of a family of oxidoreductase proteins that share the conserved active site sequence Cys-X-Y-Cys, the so-called "thioredoxin fold", a disulfide bond is formed between the two cysteines (Cys-32 and Cys-35) in the oxidized form. Although both sulfhydryl groups are solvent accessible in the reduced forms, only one of the cysteine residues (Cys-32) is involved in the adduct formation in the presence of certain alkylating agents. This disparate nucleophilicity is due to the fact that the pKa of the Cys-32 thiol group is lower than normally observed for cysteine residues by several pH units. This has been proposed to be due to the fact that the pKa of the Cys-32 thiol group is lower than normally observed for cysteine residue by several pH units. This has been proposed to be due in part to favorable interactions between this thiolate anion and the effective positive charge at the N-terminal of the alpha helix which is immediately adjacent to the active site, and electrostatic effects from the two non-cysteine residues in the "thioredoxin fold". Further stabilization of the thiolate anion can also arise from interactions with positively charged groups, such as the basic residues arginine and lysine. A recent report on the alkylation of a protein disulfide isomerase isoform and also glutathione S-transferase by the reactive trifluoroacetyl halide metabolite of the anesthesia halothane further suggests that the thioredoxin protein family may be a target for xenobiotic metabolites, and that the possible role of the active site cysteines in this alkylation mechanism should be explored. Glutathione conjugation can induce cytotoxicity by enhancing by enhancing the reactivity of xenobiotic chemicals such as the 1,2-dihaloethanes. Once activated in this manner, these glutathiones have been shown to alkylate proteins at a much higher rate than nucleic acid alkylation. We hypothesize that the active site of proteins containing the "thioredoxin fold" such as thioredoxin, thioredoxin reductase and protein disulfide isomerase are in vivo targets for alkylation by glutathione conjugates. The primary product of the alkylation of reduced E. coli thioredoxin with the rpisulfonium ion derived from S-(2-chloroethyl) glutathione has been determined to the S-(2-ethyl) glutathione adduct at Cys-32. We propose to study this S-(2-ethyl) glutathione-adducted thioredoxin via multidimensional, heteronuclear NMR experiments to elucidate the structure of the alkylated protein for comparison to the unmodified thioredoxin. 15NMR relaxation studies will allow us to compare the changes in the amplitude and timescales of motions in the S-(2-ethyl)glutathione-adducted protein's backbone with that of the unmodified protein. A mechanism in which a positively charged side chain distant from Cys-32 in the primary sequence, but close to the active site in the folded protein, may stabilize the reactive thiolate anion will then be addressed, as well as its possible role as an intermediate in protein acetylation by halothanes. The effects of thioredoxin alkylation on protein refolding will be followed by NOE experiments and hydrogen-deuterium pulse exchange techniques.